Nearly all human pancreatic adenocarcinomas (PACs) are caused by oncogenic (activating) mutations in the KRas gene. There are no treatments that reverse the actions of activated KRas mutants. The consequence of activating KRas mutations is the activation of a protein called Raf. Of the two major isoforms of Raf (C-Raf and B-Raf), C-Raf is the principle target of oncogenic Ras mutants in most cancers. A major function of KRas is to position C-Raf for activating phosphorylations. Our approach is to identify novel protein kinases that are required for KRas-dependent phosphorylation of C-Raf. There are two essential Ras-dependent phosphorylations of C-Raf, one on serine 338 (S338) and another on tyrosine 341 (Y341). These two sites within C-Raf are constitutively phosphorylated in KRas-mutant PACs, and are both required for the Ras-dependent cell proliferation. The tyrosine kinase Src is the only kinase that has been proposed to carry out the phosphorylation of Y341. A number of candidate kinases have proposed to carry out the phosphorylation of S338. We show preliminary pharmacological and molecular data challenging both of these models of C-Raf activation, and propose that both kinases have yet to be identified. We propose to identify the two kinases responsible for each of these phosphorylations by conducting biochemical screens using a panel of kinase inhibitors in a set of human PAC cell lines. We will use a functional siRNA screen to determine whether the identified S388 and Y341 kinases regulate KRas-dependent proliferation of PAC cell lines. The significance of this proposal is that the identification of novel serine and tyrosine kinases required for Ras-induced cell proliferation wil provide new therapeutic targets for inhibiting the growth of pancreatic adenocarcinomas. Despite the significance of discovering a new target for Ras-mutant cancers, this approach has not yet been attempted. The identification of new targets will contribute greatly to the field by providing new avenues for drug discovery, translational research, and ultimately novel therapeutics. Inhibition of either of these new targets will be specifically detrimental to the tumr cells, as they block signaling from oncogenic Ras to C-Raf while sparing signaling from normal Ras to B-Raf. This has the potential for increased drug tolerance and a higher therapeutic index.